The biological efficacy of interstrand crosslink (ICL)-inducing agents resides in their ability to prevent transient strand separation that is integral to DNA replication, RNA transcription, and recombination, making these bifunctional compounds effective antimicrobial and chemotherapeutic agents (Noll et al., Chem. Rev. 106:277-301, 2006; Lehoczky et al., FEMS Microbiol. Rev. 31:109-133, 2007). In addition, various endogenously generated bis-electrophiles, such as products of lipid peroxidation, are also capable of forming ICLs (Kozekov et al., J. Am. Chem. Soc. 125:50-61, 2003).
The processing and repair of ICLs in eukaryotic cells is extremely complex, potentially involving multiple DNA repair and damage tolerance pathways, including homologous recombination, nucleotide excision repair, translesion DNA synthesis, transcription-coupled repair, nonhomologous end joining, mismatch repair, cell cycle checkpoints, and ubiquitination/de-ubiquitination pathways (Noll et al., Chem. Rev. 106:277-301, 2006; Lehoczky et al., FEMS Microbiol. Rev. 31:109-133, 2007). The complexity of ICL repair and tolerance is further evident by data demonstrating that different organisms may preferentially use alternative pathways that are dependent on the stage of the cell cycle in which the ICL is encountered (Noll et al., Chem. Rev. 106:277-301, 2006; Lehoczky et al., FEMS Microbiol. Rev. 31:109-133, 2007).
Although many models for ICL repair require the involvement of homologous recombination, an alternative, recombination-independent pathway exists that utilizes endonucleases for strand incision surrounding the ICL on one of the two DNA strands, and translesion synthesis (TLS) polymerases for gap-filling replication past the ICL site on the other strand (Wang et al., Mol. Cell Biol. 21:713-720, 2001; Zheng et al., Mol. Cell Biol. 23:754-761, 2003; Richards et al., Nucleic Acids Res. 33:5382-5393, 2005; Sarkar et al., EMBO J. 25, 1285-1294, 2006; Shen et al., J. Biol. Chem. 281:13869-13872, 2006; Liu et al., Biochemistry 45:12898-12905, 2006). In these repair models, the dually-incised strand possesses sufficient mobility that a DNA polymerase can strand displace the nucleotide patch that is 5′ to the lesion, then replicate past the ICL site to complete the repair gap-filling synthesis.
Insights into the essential genes for ICL repair and mutagenesis in Saccharomyces cerevisiae demonstrate a role for the product of rev3, the catalytic subunit of pol ζ (Henriques and Moustacchi, Genetics 95:273-288, 1980; Grossmann et al., Mutat. Res. 487:73-83, 2001). Further support for involvement of pol and another TLS polymerase, Rev1, in tolerance to ICL damage and their contribution to ICL-associated mutagenesis was obtained in both yeast (Sarkar et al., EMBO J. 25, 1285-1294, 2006; Wu et al., Cancer Res. 64:3940-3948, 2004) and vertebrate cells (Richards et al., Nucleic Acids Res. 33:5382-5393, 2005; Shen et al., J. Biol. Chem. 281:13869-13872, 2006; Nojima, et al., Cancer Res. 65:11704-11711, 2005).
Previous models for ICL repair have postulated the following sequential steps leading to restoration of an intact DNA strand: (i) DNA strand incision by components of nucleotide excision repair (NER); (ii) failure of pol ε to catalyze gap filling; (iii) monoubiquitination of proliferating cell nuclear antigen (PCNA); and (iv) recruitment of a TLS polymerase to replicate past the lesion and fill the gap (Sarkar et al., EMBO J. 25, 1285-1294, 2006). Despite previous investigations into the mechanisms of ICL repair, there remains a need to identify the specific enzymes involved in this type of repair process to improve treatments with ICL-inducing agents.